The primary goal of the proposed studies is to dissect the molecular mechanism of nonself recognition in filamentous fungi using Neurospora crassa as a model system. Nonself recognition leads to a type of fungal programmed cell death (PCD) termed heterokaryon incompatibility (HI). HI is ubiquitous in filamentous fungi and can be likened to a fungal immune system, which confers selective advantages by preventing hyphal fusion events that spread mycoviruses, debilitated organelles and deleterious plasmids throughout a fungal population. Nonself recognition during vegetative growth in filamentous fungi is mediated by genetic loci, termed het (for heterokaryon). Similar to other nonself recognition loci, such as the Major Histocompatibility Complex (MHC) in jawed vertebrates, het loci in N. crassa show evidence of balancing selection. From our previous funding period, we identified a new protein required for nonself recognition and death, called PIN-C, which contains a fungal-specific death effector domain, called HET. We determined that cell death through HI does not occur via an apoptotic mechanism, although reactive oxygen species are an early event associated with nonself recognition. We also identified a transcription factor called vib-1 (vegetative incompatibility blocked) that is required both for cell death and for proper secretion. Finally, we have very recent data indicating that the genes that regulate nonself recognition interfere with signaling required for chemotropic interactions and cell fusion, thus indicating an intriguing link between signaling/machinery associated with chemotropism/cell fusion and nonself recognition proteins and cell death. In this proposal, we describe experiments to further dissect the molecular mechanism of nonself recognition, unravel the pathway to death and identify new nonself recognition loci using genomics approaches. Finally, we describe experiments to examine the exciting connection between cell signaling involved in fusion and nonself recognition, an aspect that we recently determined occurs at a distance. We believe that a dissection of fungal PCD using N. crassa as a model organism is an attractive fungal-specific target for the development of drugs to combat human fungal infections and provides a fascinating comparative model for evolutionary studies on nonself recognition loci.